Figure 2. Identification of Hydrogen Bonds in E4B U-box by NMR Spectroscopy.

(A) A region from the constant time 3D J-HNCO NMR spectrum of E4B U-box showing peaks correlated through ¹J_NC’^{, 2}J_NC’^{, 3}J_NCγ and ^h3J_NC’ scalar couplings.

(B) The nineteen H-bonds in E4B U-box that are listed in Table S3 were identified through ^h3J_NC’ scalar coupling measurements. The dotted lines illustrate these H-bonds. Residues Asp1232 and Asp1268 for which side chain ^h3J_NC’ scalar couplings were measured are shown. Asp1232 and Asp1268 correspond to the zinc binding regions of RING domains.

(C) Asp1232 and Asp1268 of E4B U-box approximately occupy the zinc centers (purple spheres) in c-Cbl RING domain (PDB entry 1FBV) when the U-box and RING domains are superimposed. H-bonds involving the two aspartate side chains were determined from ^h3J_NC’ scalar coupling measurements and are shown by the dashed lines.

(D) Calibration curve for the dihedral angle (χ₁) dependence of ³J_NCγ scalar couplings in aspartate and asparagine residues. Blue and red points correlate experimental ³J_NCγ and corresponding χ₁ angle values from the crystal structure of E4B U-box. The red points correspond to residues for which a side chain carbonyl participates in a H-bond as determined from ^h3J_NC scalar couplings.

See also Table S1 for the values of measured coupling constants.